3.2.10. Bromelain Activity

The bromelain assay was established according to Chakraborty et al. (2014) [60] with some modifications. The reaction involved 50 μL enzyme extract (from PBP) and 1150 μL casein 1% (w/v) (Sigma–Aldrich, Germany) solution in glycine 0.1 mol·L⁻¹ (Sigma–Aldrich, Germany) and 25 mmol·L⁻¹ cysteine (Sigma–Aldrich, Germany). The mixture was incubated in a shaking water bath (10 min at 37 °C) and the reaction was stopped by adding 1.8 mL trichloroacetic acid 5% (w/v). The assay mixture was filtrated with a syringe filter (0.45 μm) and the absorbance measurement was performed at 280 nm. The bromelain activity was calculated using a tyrosine (Alfa Aesar, Lancashire, UK) standard curve and expressed as the amount of tyrosine on a dry weight basis reported as μmol tyrosine. min⁻¹·g⁻¹ dry weight.